Many eukaryotic genes are regulated in an inducible, cell type-specific fashion. Genes expressed in response to heat shock, steroid/thyroid hormones, phorbol esters, cyclic adenosine monophosphate (cAMP), growth factors and heavy metal ions are examples of this class. The activity of cells is controlled by external signals that stimulate or inhibit intracellular events. The process by which an external signal is transmitted into and within a cell to elicit an intracellular response is referred to as signal transduction. Signal transduction is generally initiated by the interaction of extracellular factors (or inducer molecules, i.e., growth factors, hormones, adhesion molecules, neurotransmitters, and other mitogens) with receptors at the cell surface. Extracellular signals are transduced to the inner face of the cell membrane, where the cytoplasmic domains of receptor molecules contact intracellular targets. The initial receptor-target interactions stimulate a cascade of additional molecular interactions involving multiple intracellular pathways that disseminate the signal throughout the cell.
Many of the proteins involved in signal transduction contain multiple domains. Some of these domains have enzymatic activity and some of these domains are capable of binding to other cellular proteins, DNA regulatory elements, calcium, nucleotides, lipid mediators, and the like.
Protein—protein interactions are involved in all stages of the intracellular signal transduction process—at the cell membrane, where the signal is initiated in the cytoplasm by receptor recruitment of other cellular proteins, in the cytoplasm where the signals are disseminated to different cellular locations, and in the nucleus where proteins involved in transcriptional control congregate to turn on or turn off gene expression.
Mitogenic signaling affects the transcriptional activation of specific sets of genes and the inactivation of others. The nuclear effectors of gene activation are transcription factors that bind to DNA as homomeric or heteromeric dimers. Phosphorylation also modulates the function of transcription factors, as well. Oncogenes, first identified as the acute transforming genes transduced by retroviruses, are a group of dominantly acting genes. Such genes, which are involved in cell division, encode growth factors and their receptors, as well as second messengers and mitogenic nuclear proteins activated by growth factors.
The binding of growth factors to their respective receptors activates a cascade of intracellular pathways that regulate phospholipid metabolism, arachidonate metabolism, protein phosphorylation, calcium mobilization and transport, and transcriptional regulation. Specific phosphorylation events mediated by protein kinases and phosphatases modulate the activity of a variety of transcription factors within the cell. These signaling events can induce changes in cell shape, mobility, and adhesiveness, or stimulate DNA synthesis. Aberrations in these signal-induced events are associated with a variety of hyperproliferative diseases ranging from cancer to psoriasis.
The ability to repress intracellular signal-induced response pathways is an important mechanism in negative control of gene expression. Selective disruption of such pathways would allow the development of therapeutic agents capable of treating a variety of disease states related to improper activation and/or expression of specific transcription factors. For example, in patients with non-insulin dependent diabetes mellitus (NIDDM), hyperglycemia develops, in part as a result of β cell failure secondary to chronic insulin resistance. This hyperglycemia appears to be exacerbated by hyperglucogonemia and increased hepatic gluconeogenesis. cAMP appears to be the major starvation state signal which triggers glucagon gene expression as well as transcription of PEPCK, the rate limiting enzyme in gluconeogenesis.
Hyperglycemia is associated with an increased risk for all of the common late complications of diabetes mellitus, which are the major causes of morbidity and mortality in diabetics. However, there is no generally applicable and consistently effective means of maintaining plasma glucose fluctuations within a normal range in diabetics, and efforts to do so entail significant risks of causing frequent or severe hypoglycemic episodes. Nevertheless, common treatments include diet management and the use of insulin preparations and oral hypoglycemic agents.
Diabetes mellitus is among the most common of all metabolic disorders, affecting up to 11% of the population by age 70. Type I diabetes (insulin dependent diabetes mellitus or IDDM) represents about 5 to 10% of this group and is the result of a progressive autoimmune destruction of the pancreatic beta-cells with subsequent insulin deficiency.
There are two classes of type II diabetes (non-insulin dependent diabetes mellitus or NIDDM). One typically presents in older people; thus it is sometimes called mature onset diabetes. Another form, though similar to mature onset, presents in a subject at a very early age. Type II diabetes represents 90–95% of the affected population, more than 100 million people worldwide (King, H. and Zimmer, P. (1988) Wld Hlth. Statist. Quart. 41:190–196; Harris, M. I., et al. (1992) Diabetes Care 15:815–819), and is associated with peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion (DeFronzo, R. A. (1988) Diabetes 37:667–687). Family studies point to a major genetic component (Newman, B. et al. (1987) Diabetologia 30:763–768; Kobberling, J. (1971) Diabetologia 7:46–49; Cook, J. T. E. (1994) Diabetologia 37:1231–1240). However, few susceptibility genes have been identified.
Type II diabetes is characterized by a patient's inability to respond to insulin and/or insufficient insulin secretion. Insulin exerts a dominant effect on the regulation of glucose homeostasis. In the liver, insulin inhibits the production of glucose by inhibiting gluconeogenesis and glycogenolysis. Insulin is thought to act by causing cells to absorb glucose from the blood stream. Once absorbed, the liver converts glucose to glycogen. The liver supplies glucose by converting glycogen stores to glucose. Insulin also has a major role in the regulation of protein and lipid metabolism through a variety of actions that affect the flux of protein and lipid substrates.
The glucolytic pathway is central in the understanding of the lack of glucose homeostasis in diabetes. There is very tight cellular control of glycolysis. This control is achieved by the regulatory inhibition by certain glycolytic enzymes depending on the level of various glucose degradation products. Since the degradation products of glucose are also important precursors or intermediates in other aspects of metabolism, the regulatory enzymes in carbohydrate catabolism also recognize and respond to appropriate signals from other metabolic pathways.
Some of the enzymes involved in the degradation of glucose are known to be regulated and/or contain binding sites for HNF1 and/or HNF4. The first step in the degradation of glucose is the phosphorylation of glucose to glucose-6-phosphate and is carried out by an enzyme called glucokinase. The hormone insulin, secreted by the pancreas into the blood whenever the blood glucose concentration is high, stimulates the synthesis of glucokinase. In the diabetic condition, which is characterized by a defect in insulin secretion and/or amount of insulin, glucokinase is typically also deficient.
In addition to regulation of the rate of glycolysis through control of the entry of free glucose, the sequence of reactions from glucose to pyruvate is also under biological control. The enzyme that converts phosphoenol pyruvate to pyruvate and ATP is called pyruvate kinase. At high ATP concentrations the affinity of pyruvate kinase for phosphoenol pyruvate is relatively low. Pyruvate kinase is also inhibited by acetyl-CoA and by long chain fatty acids. Therefore, whenever ample fuels are available for respiration, glycolysis is inhibited by the action of pyruvate kinase. Since glucokinase contains HNF1 binding sites at its promoter region and pyruvate kinase contains HNF4 binding sites, modulation of the bioactivity of glucokinase and/or pyruvate kinase can be an effective therapy for type II diabetes.
As mentioned above, insulin has a role in lipid metabolism. Diabetes is associated with lipid related disorders such as obesity, elevated cholesterol and triglycerides. When there is an excess of glucose in the blood, the liver produces lipids instead of glucose. High levels of circulating insulin causes the liver to increase the production of lipids. Therefore, metabolism of lipids and carbohydrates is intimately related.
There remains, therefore, a need in the art for methods to selectively disrupt intracellular signal-induced response pathways. In addition, there remains a need in the art for more effective methods for the treatment of diabetes and for detecting predisposed individuals so as to provide pre-emptive therapy early in the disease or to detect early stage disease so as to reduce morbidity.